Paneth cells are the primary source of C-type lysozyme, a b-1,4-N-acetylmuramoylhydrolase that enzymatically processes bacterial cell walls. Paneth cells are normally present in human cecum and ascending colon, but are rarely found in descending colon and rectum; Paneth cell metaplasia in this region and aberrant lysozyme production are hallmarks of inflammatory bowel disease (IBD) pathology. Here, we examined the impact of aberrant lysozyme production in colonic inflammation. Targeted disruption of Paneth cell lysozyme (Lyz1) protected mice from experimental colitis. Lyz1-deficiency diminished intestinal immune responses to bacterial molecular patterns and resulted in the expansion of lysozyme-sensitive mucolytic bacteria, including Ruminococcus gnavus, a Crohn's disease-associated pathobiont. Ectopic lysozyme production in colonic epithelium suppressed lysozyme-sensitive bacteria and exacerbated colitis. Transfer of R. gnavus into Lyz1 À/À hosts elicited a type 2 immune response, causing epithelial reprograming and enhanced anti-colitogenic capacity. In contrast, in lysozyme-intact hosts, processed R. gnavus drove pro-inflammatory responses. Thus, Paneth cell lysozyme balances intestinal anti-and pro-inflammatory responses, with implications for IBD.

Background Intestinal microbiota influences the progression of colitis-associated colorectal cancer (CAC). With diet being a key determinant of the gut microbial ecology, dietary interventions are an attractive avenue for the prevention of CAC. Curcumin is the most active constituent of the ground rhizome of the Curcuma Longa plant, which has been demonstrated to have anti-inflammatory, anti-oxidative and anti-proliferative properties. Methods Il10−/− mice on 129/SvEv background were used as a model of CAC. Starting at 10 weeks of age, WT or Il10−/− mice received six weekly i.p. injections of azoxymethane (AOM) or saline, and were started on either a control or curcumin-supplemented diet. Stools were collected every 4 weeks for microbial community analysis. Mice were sacrificed at 30 weeks of age. Results Curcumin-supplemented diet increased survival, decreased colon weight/length ratio, and at 0.5%, entirely eliminated tumor burden. Although colonic histology indicated improvement with curcumin, no effects of mucosal immune responses have been observed in PBS/Il10−/− mice, and limited effects were seen in AOM/Il10−/− mice. In WT and in Il10−/− mice, curcumin increased bacterial richness, prevented age-related decrease in alpha diversity, increased the relative abundance of Lactobacillales, and decreased Coriobacterales order. Taxonomic profile of AOM/Il10−/− mice receiving curcumin was more similar to those of wild-type mice than those fed control diet. Conclusions In AOM/Il10−/− model, curcumin reduced or eliminated colonic tumor burden with limited effects on mucosal immune responses. The beneficial effect of curcumin on tumorigenesis was associated with the maintenance of a more diverse colonic microbial ecology.

Background & Aims Klotho deficiency in hypomorphic KL mice leads to premature senescence and phenotype consistent with impaired mineral homeostasis. Klotho has anti-inflammatory properties protecting from NO-induced endothelial dysfunction, reduces the expression of endothelial adhesion molecules, and may contribute to T-cell dysfunction. Since defective Ca2+/Pi homeostasis leading to osteopenia/osteoporosis is frequently associated with human IBD, we investigated the changes in Klotho gene expression as a consequence of experimental colitis. Methods We utilized three murine IBD models: TNBS colitis, microflora-induced colitis in gnotobiotic IL-10−/− mice, and adoptive CD4+CD45RBhigh T-cell transfer colitis. These studies were followed by in vitro approaches using renal epithelial cells (mpkDCT4 and mIMCD3), and the cloned murine KL gene promoter. Results Renal expression of Klotho mRNA and protein was significantly inhibited in all three models of human IBD. This degree of inhibition was correlated with the severity of colitis, and was reversed by neutralizing anti-TNF antibodies. In vitro, TNF resulted in a significant inhibition of KL expression and was further potentiated by IFN-γ. TNF/IFN-γ combination resulted in increased iNOS expression and significantly elevated the concentration of NO in medium. The effect of IFN-γ could be reproduced by cell exposure to SNAP (NO donor), and reversed by iNOS inhibitor, L-NIL. The cytokine effects were transcriptionally mediated since Klotho mRNA stability remained unaffected, while reporter constructs with the mKL gene promoter displayed significant downregulation in transiently transfected renal epithelial cells. Conclusions These novel findings could help explain several extraintestinal complications including abnormalities in bone homeostasis in patients with chronic colitis.

Na+/H+ exchanger 3 (NHE3) provides a major route for intestinal Na+ absorption. NHE3 has been considered a target of proinflammatory cytokines and enteropathogenic bacteria, and impaired NHE3 expression and/or activity may be responsible for inflammation-associated diarrhea. However, the possibility of loss of NHE3 function reciprocally affecting gut immune homeostasis has not been investigated. In this report, we describe that NHE3-deficient mice spontaneously develop colitis restricted to distal colonic mucosa. NHE3(-/-) mice housed in a conventional facility exhibited phenotypic features such as mild diarrhea, occasional rectal prolapse, and reduced body weight. Genomewide microarray analysis identified not only a large group of transport genes that potentially represent an adaptive response, but also a considerable number of genes consistent with an inflammatory response. Histological examination demonstrated changes in the distal colon consistent with active inflammation, including crypt hyperplasia with an increased number of 5-bromo-2'-deoxyuridine-positive cells, diffuse neutrophilic infiltrate with concomitant 15-fold increase in matrix metalloproteinase 8 expression, an increased number of pSer276-RelA-positive cells, and a significant decrease in periodic acid-Schiff-positive goblet cells. Real-time PCR demonstrated elevated expression of inducible nitric oxide synthase (38-fold), TNF-alpha (6-fold), macrophage inflammatory protein-2 (48-fold), and IL-18 (3-fold) in the distal colon of NHE3(-/-) mice. NHE3(-/-) mice showed enhanced bacterial adhesion and translocation in the distal colon. Colitis was ameliorated by oral administration of broad-spectrum antibiotics. In conclusion, NHE3 deficiency leads to an exacerbated innate immune response, an observation suggesting a potentially novel role of NHE3 as a modifier gene, which when downregulated during infectious or chronic colitis may modulate the extent and severity of colonic inflammation.

In vitrodata and transgenic mouse models suggest a role for TGFβ signaling in dendritic cells (DC) to prevent autoimmunity primarily through maintenance of DCs in their immature and tolerogenic state characterized by low expression of MHCII and co-stimulatory molecules, and increased expression of indoleamine 2,3-dioxygenase (IDO), among others. To test whether a complete lack of TGFβ signaling in DCs predisposes mice to spontaneous autoimmunity, and to verify the mechanisms implicated previously in vitro, we generated conditional knock-out mice with Cre-mediated DC-specific deletion of Tgfbr2 (DC-Tgfbr2 KO). DC-Tgfbr2 KO mice die before 15 weeks of age with multi-organ autoimmune inflammation and spontaneous activation of T and B cells. Interestingly, there were no significant differences in the expression of MHCII, co-stimulatory molecules, or IDO in secondary lymphoid organ DCs, although Tgfbr2-deficient DCs were more pro-inflammatory in vitro and in vivo. DC-Tgfbr2 KO showed attenuated FoxP3 expression in regulatory T cells (Tregs) and abnormal expansion of CD25−FoxP3+ Tregs in vivo. Tgfbr2-deficient DCs secreted elevated levels of IFNγ and were not capable of directing antigen-specific Treg conversion unless in the presence of anti-IFNγ blocking antibody. Adoptive transfer of iTregs into DC-Tgfbr2 KO mice partially rescued the phenotype. Therefore, in vivo, TGFβ signaling in DCs is critical in the control of autoimmunity through both Treg dependent and independent mechanisms, but it does not affect MHCII and co-stimulatory molecule expression.